RNA viruses cause widespread disease in humans and have significant medical, economic, and social repercussions. The overall objective of this application is to provide detailed insight into how rotaviruses, RNA viruses that cause severe gastroenteritis in children, transcribe their genomes in the context of intact, subviral, double-layered particles. While high-resolution structures exist for non-transcribing rotavirus double-layered particles, little is known about the structure of particles undergoing mRNA synthesis. The central hypothesis is that the rotavirus double-layered particle undergoes dynamic structural rearrangements during the process of transcription. This hypothesis will be tested through two integrated, yet independent, specific aims: (i) determine the three-dimensional structures of actively-transcribing rotavirus double-layered particles to better than 10-A resolution using cryo-electron miscroscopy and (ii) determine the three-dimensional structures of actively-transcribing rotavirus double-layered particles to better than 20-A resolution using a novel liquid imaging platform called in situ molecular microscopy. Specifically, transcriptionally-competent double-layered particles will be isolated from rotavirus-infected cells, induced to perform mRNA synthesis in vitro, and then either flash-frozen in vitreous ice or submerged in a microfluidic chamber prior to being imaged using a transmission electron microscope. State-of-the-art image processing software that relies on Bayesian inference will be employed to derive three-dimensional reconstructions of double-layered particles in ice or liquid. This proposal is innovative because it applies new technologies to investigate the structure of the enzymatically-active rotavirus transcriptase complex for which little information currently exists. The work is significant because it is expected to be the first step in a continuum of research aimed at developing pharmacological strategies to obliterate RNA virus transcription. Equally important, this work will advance our technical capabilities to visualize biological assemblies in liquid, thereby bringing us one step closer to 'live' EM imaging.